Heat stable mutants of starch biosynthesis enzymes

ABSTRACT

The subject invention pertains to novel mutant polynucleotide molecules that encode enzymes that have increased heat stability. These polynucleotides, when expressed in plants, result in increased yield in plants grown under conditions of heat stress. In one embodiment, the polynucleotide molecules of the subject invention encode maize endosperm ADP glucose pyrophosphorylase (AGP) and soluble starch synthase (SSS) enzyme activities. Plants and plant tissue bred to contain, or transformed with, the mutant polynucleotides, and expressing the polypeptides encoded by the polynucleotides, are also contemplated by the present invention. The subject invention also concerns methods for isolating polynucleotides and polypeptides contemplated within the scope of the invention. Methods for increasing yield in plants grown under conditions of heat stress are also provided.

CROSS-REFERENCE TO RELATED APPLICATION This application claims thebenefit of U.S. Provisional application Serial No. 60/275,768, filedMar. 14, 2001.

[0001] This invention was made with government support under NationalScience Foundation grant number 9316887. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

[0002] The sessile nature of plant life generates a constant exposure toenvironmental factors that exert positive and negative effects on itsgrowth and development. One of the major impediments facing modemagriculture is adverse environmental conditions. One important factorwhich causes significant crop loss is heat stress. Temperature stressgreatly reduces grain yield in many cereal crops such as maize, wheat,and barley. Yield decreases due to heat stress range from 7 to 35% inthe cereals of world-wide importance.

[0003] A number of studies have identified likely physiologicalconsequences of heat stress. Early work by Hunter et al. (Hunter, R. B.,Tollenaar, M., and Breuer, C. M. [1977] Can. J. Plant Sci. 57:1127-1133)using growth chamber conditions showed that temperature decreased theduration of grain filling in maize. Similar results in which theduration of grain filling was adversely altered by increasedtemperatures were identified by Tollenaar and Bruulsema (Tollenaar, M.and Bruulsema, T. W. [1988] Can. J. Plant Sci. 68:935-940). Badu-Aprakuet al. (Badu-Apraku, B., Hunter, R. B., and Tollenaar, M. [1983] Can. J.Plant. Sci. 63:357-363) measured a marked reduction in the yield ofmaize plants grown under the day/night temperature regime of 35/15° C.compared to growth in a 25/15° C. temperature regime. Reduced yields dueto increased temperatures is also supported by historical as well asclimatological studies (Thompson, L. M. [1986] Agron. J. 78:649-653;Thompson, L. M. [1975] Science 188:535-541; Chang, J. [1981] Agricul.Metero. 24:253-262; and Conroy, J. P., Seneweera, S., Basra, A. S.,Rogers, G., and Nissen-Wooller, B. [1994] Aust. J. Plant Physiol.21:741-758).

[0004] That the physiological processes of the developing seed areadversely affected by heat stress is evident from studies using an invitro kernel culture system (Jones, R. J., Gengenbach, B. G., andCardwell, V. B. [1981] Crop Science 21:761-766; Jones, R. J., Ouattar,S., and Crookston, R. K. [1984] Crop Science 24:133-137; and Cheikh, N.,and Jones, R. J. [995] Physiol. Plant. 95:59-66). Maize kernels culturedat the above-optimum temperature of 35° C. exhibited a dramaticreduction in weight.

[0005] Work with wheat identified the loss of soluble starch synthase(SSS) activity as a hallmark of the wheat endosperm's response to heatstress (Hawker, J. S. and Jenner, C. F. [1993] Aust. J. Plant Physiol.20:197-209; Denyer, K., Hylton, C. M., and Smith, A. M. [1994] Aust. J.Plant Physiol. 21:783-789; Jenner, C. F. [1994] Aust. J. Plant Physiol.21:791-806). Additional studies with SSS of wheat endosperm show that itis heat labile (Rijven, A. H. G. C. [1986] Plant Physiol. 81:448-453;Keeling, P. L., Bacon, P. J., Holt, D. C. [1993] Planta. 191:342-348;Jenner, C. F., Denyer, K., and Guerin, J. [1995] Aust. J. Plant Physiol.22:703-709).

[0006] The roles of SSS and ADP glucose pyrophosphorylase (AGP) underheat stress conditions in maize is less clear. AGP catalyzes theconversion of ATP and α-glucose-1-phosphate to ADP-glucose andpyrophosphate. ADP-glucose is used as a glycosyl donor in starchbiosynthesis by plants and in glycogen biosynthesis by bacteria. Theimportance of ADP-glucose pyrophosphorylase as a key enzyme in theregulation of starch biosynthesis was noted in the study of starchdeficient mutants of maize (Zea mays) endosperm (Tsai, C. Y., andNelson, Jr., O. E. [1966] Science 151:341-343; Dickinson, D. B., J.Preiss [1969] Plant Physiol. 44:1058-1062).

[0007] Ou-Lee and Setter (Ou-Lee, T. and Setter, T.L. [1985] PlantPhysiol. 79:852-855) examined the effects of temperature on the apicalor tip regions of maize ears. With elevated temperatures, AGP activitywas lower in apical kernels when compared to basal kernels during thetime of intense starch deposition. In contrast, in kernels developed atnormal temperatures, AGP activity was similar in apical and basalkernels during this period. However, starch synthase activity duringthis period was not differentially affected in apical and basal kernels.Further, heat-treated apical kernels exhibited an increase in starchsynthase activity over control. This was not observed with AGP activity.Singletary et al. (Singletary, G. W., Banisadr, R., and Keeling, P. L.[1993] Plant Physiol. 102: 6 (suppl).; Singletary, G. W., Banisadra, R.,Keeling, P. L. [1994] Aust. J. Plant Physiol. 21:829-841) using an invitro culture system quantified the effect of various temperaturesduring the grain fill period. Seed weight decreased steadily astemperature increased from 22-36° C. A role for AGP in yield loss isalso supported by work from Duke and Doehlert (Duke, E. R. and Doehlert,D. C. [1996] Environ. Exp. Botany. 36:199-208).

[0008] Work by Keeling et al. (1994, supra) quantified SSS activity inmaize and wheat using Q₁₀ analysis, and showed that SSS is an importantcontrol point in the flux of carbon into starch.

[0009] In vitro biochemical studies with AGP and SSS clearly show thatboth enzymes are heat labile. Maize endosperm AGP loses 96% of itsactivity when heated at 57° C. for five minutes (Hannah, L. C.,Tuschall, D. M., and Mans, R. J. [1980] Genetics 95:961-970). This is incontrast to potato AGP which is fully stable at 70° C. (Sowokinos, J. R.and Preiss, J. [1982] Plant Physiol. 69:1459-1466; Okita, T. W., Nakata,P. A., Anderson, J. M., Sowokinos, J., Morell, J., and Preiss, J. [1990]Plant Physiol. 93:785-90). Heat inactivation studies with SSS showedthat it is also labile at higher temperatures, and kinetic studiesdetermined that the Km value for amylopectin rose exponentially whentemperature increased from 25-45° C. (Jenner et al., 1995, supra).

[0010] Biochemical and genetic evidence has identified AGP as a keyenzyme in starch biosynthesis in higher plants and glycogen biosynthesisin E. coli (Preiss, J. and Romeo, T. [1994] Progress in Nuc. Acid Res.and Mol Biol. 47:299-329; Preiss, J. and Sivak, M. [1996] “Starchsynthesis in sinks and sources,” In Photoassimilate distribution inplants and crops: source-sink relationships. Zamski, E., ed., MarcilDekker Inc. pp. 139-168). AGP catalyzes what is viewed as the initialstep in the starch biosynthetic pathway with the product of the reactionbeing the activated glucosyl donor, ADPglucose. This is utilized bystarch synthase for extension of the polysaccharide polymer (reviewed inHannah, L. Curtis [1996] “Starch synthesis in the maize endosperm,” In:Advances in Cellular and Molecular Biology of Plants, Vol. 4. B. A.Larkins and I. K. Vasil (eds.). Cellular and Molecular Biology of PlantSeed Development. Kluwer Academic Publishers, Dordrecht, TheNetherlands).

[0011] Initial studies with potato AGP showed that expression in E. coliyielded an enzyme with allosteric and kinetic properties very similar tothe native tuber enzyme (Iglesias, A., Barry, G. F., Meyer, C.,Bloksberg, L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W.,Kishore, G. M., and Preiss, J. [1993] J. Biol Chem. 268:1081-86;Ballicora, M. A., Laughlin, M. J., Fu, Y., Okita, T. W., Barry, G. F.,and Preiss, J. [1995] Plant Physiol. 109:245-251). Greene et al.(Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J.,and Okita, T. W. [1996] Proc. Natl. Acad. Sci. 93:1509-1513; Greene, T.W., Woodbury, R. L., and Okita, T. W. [1996] Plant Physiol.(112:1315-1320) showed the usefulness of the bacterial expression systemin their structure-function studies with the potato AGP. Multiplemutations important in mapping allosteric and substrate binding siteswere identified (Okita, T. W., Greene, T. W., Laughlin, M. J., Salamone,P., Woodbury, R., Choi, S., Ito, H., Kavakli, H., and Stephens, K.[1996] “Engineering Plant Starches by the Generation of Modified PlantBiosynthetic Enzymes,” In Engineering Crops for Industrial End Uses,Shewry, P. R., Napier, J. A., and Davis, P., eds., Portland Press Ltd.,London).

[0012] AGP enzymes have been isolated from both bacteria and plants.Bacterial AGP consists of a homotetramer, while plant AGP fromphotosynthetic and non-photosynthetic tissues is a heterotetramercomposed of two different subunits. The plant enzyme is encoded by twodifferent genes, with one subunit being larger than the other. Thisfeature has been noted in a number of plants. The AGP subunits inspinach leaf have molecular weights of 54 kDa and 51 kDa, as estimatedby SDS-PAGE. Both subunits are immunoreactive with antibody raisedagainst purified AGP from spinach leaves (Copeland, L., J. Preiss (1981)Plant Physiol. 68:996-1001; Morell, M., M. Bloon, V. Knowles, J. Preiss[1988] J. Bio. Chem. 263:633). Immunological analysis using antiserumprepared against the small and large subunits of spinach leaf showedthat potato tuber AGP is also encoded by two genes (Okita et al., 1990,supra). The cDNA clones of the two subunits of potato tuber (50 and 51kDa) have also been isolated and sequenced (Muller-Rober, B. T., J.Kossmann, L. C. Hannah, L. Willmitzer, U. Sounewald [1990] Mol. Gen.Genet. 224:136-146; Nakata, P. A., T. W. Greene, J. M. Anderson, B. J.Smith-White, T. W. Okita, J. Preiss [1991] Plant Mol. Biol.17:1089-1093). The large subunit of potato tuber AGP is heat stable(Nakata et al. [1991], supra).

[0013] As Hannah and Nelson (Hannah, L. C., O. E. Nelson (1975) PlantPhysiol. 55:297-302.; Hannah, L. C., and Nelson, Jr., O. E. [1976]Biochem. Genet. 14:547-560) postulated, both Shrunken-2 (Sh2) (Bhave, M.R., S. Lawrence, C. Barton, L. C. Hannah [1990] Plant Cell 2:581-588)and Brittle-2 (Bt2) (Bae, J. M., M. Giroux, L. C. Hannah [1990] Maydica35:317-322) are structural genes of maize endosperm ADP-glucosepyrophosphorylase. Sh2 and Bt2 encode the large subunit and smallsubunit of the enzyme, respectively. From cDNA sequencing, Sh2 and Bt2proteins have predicted molecular weight of 57,179 Da (Shaw, J. R., L.C. Hannah [1992] Plant Physiol. 98:1214-1216) and 52,224 Da,respectively. The endosperm is the site of most starch deposition duringkernel development in maize. Sh2 and bt2 maize endosperm mutants havegreatly reduced starch levels corresponding to deficient levels of AGPactivity. Mutations of either gene have been shown to reduce AGPactivity by about 95% (Tsai and Nelson, 1966, supra; Dickinson andPreiss, 1969, supra). Furthermore, it has been observed that enzymaticactivities increase with the dosage of functional wild type Sh2 and Bt2alleles, whereas mutant enzymes have altered kinetic properties. AGP isthe rate limiting step in starch biosynthesis in plants. Stark et al.placed a mutant form of E. coli AGP in potato tuber and obtained a 35%increase in starch content (Stark et al. [1992] Science 258:287).

[0014] The cloning and characterization of the genes encoding the AGPenzyme subunits have been reported for various plants. These include Sh2cDNA (Bhave et al., 1990, supra), Sh2 genomic DNA (Shaw and Hannah,1992, supra), and Bt2 cDNA (Bae et al., 1990, supra) from maize; smallsubunit cDNA (Anderson, J. M., J. Hnilo, R. Larson, T. W. Okita, M.Morell, J. Preiss [1989] J. Biol. Chem. 264:12238-12242) and genomic DNA(Anderson, J. M., R. Larson, D. Landencia, W. T. Kim, D. Morrow, T. W.Okita, J. Preiss [1991] Gene 97:199-205) from rice; and small and largesubunit cDNAs from spinach leaf (Morell et al., 1988, supra) and potatotuber (Muller-Rober et al., 1990, supra; Nakata, P. A., Greene, T. W.,Anderson, J. W., Smith-White, B. J., Okita, T. W., and Preiss, J. [1991]Plant Mol. Biol. 17:1089-1093). In addition, cDNA clones have beenisolated from wheat endosperm and leaf tissue (Olive, M. R., R. J.Ellis, W. W. Schuch [1989] Plant Physiol. Mol. Biol. 12:525-538) andArabidopsis thaliana leaf (Lin, T., Caspar, T., Sommerville, C. R., andPreiss, J. [1988] Plant Physiol. 88:1175-1181). AGP sequences frombarley have also been described in Ainsworth et al. (Ainsworth, C.,Hosein, F., Tarvis, M., Weir, F., Burrell, M., Devos, K. M., Gale, M. D.[1995] Planta 197:1-10).

[0015] AGP functions as an allosteric enzyme in all tissues andorganisms investigated to date. The allosteric properties of AGP werefirst shown to be important in E. coli. A glycogen-overproducing E. colimutant was isolated and the mutation mapped to the structural gene forAGP, designated as glyc. The mutant E. coli, known as glyc-16, was shownto be more sensitive to the activator, fructose 1,6 bisphosphate, andless sensitive to the inhibitor, cAMP (Preiss, J. [1984] Ann. Rev.Microbiol. 419-458). Although plant AGP's are also allosteric, theyrespond to different effector molecules than bacterial AGP'S. In plants,3-phosphoglyceric acid (3-PGA) functions as an activator while phosphate(PO₄) serves as an inhibitor (Dickinson and Preiss, 1969, supra).

[0016] Using an in vivo mutagenesis system created by the Ac-mediatedexcision of a Ds transposable element fortuitously located close to aknown activator binding site, Giroux et al. (Giroux, M. J., Shaw, J.,Barry, G., Cobb, G. B., Greene, T., Okita, T. W., and Hannah, L. C.[1996] Proc. Natl. Acad. Sci. 93:5824-5829) were able to generatesite-specific mutants in a functionally important region of maizeendosperm AGP. One mutant, Rev6, contained a tyrosine-serine insert inthe large subunit of AGP and conditioned a 11-18% increase in seedweight. In addition, published international application WO 01/64928teaches that various characteristics, such as seed number, plantbiomass, Harvest Index etc., can be increased in plants transformed witha polynucleotide encoding a large subunit of maize AGP containing theRev6 mutation.

[0017] Published international patent applications WO 99/58698 and WO98/22601 and issued U.S. Pat. No. 6,069,300 disclose mutations in thelarge subunit of maize AGP enzyme that, when expressed, confersincreased heat stability in comparison to that observed for wild typeAGP enzyme. Several heat stable mutants are disclosed in the '300 patentand WO publications, including mutants designated as HS 13 (having anAla to Pro substitution at position 177); HS 14 (having an Asp to Hissubstitution at position 400 and a Val to Ile substitution at position454; HS 16 (having an Arg to Thr substitution at position 104); HS 33(having a His to Tyr substitution at position 333); HS 39 (having a Histo Tyr substitution at position 333); HS 40 (having a His to Tyrsubstitution at position 333 and a Thr to Ile substitution at position460); HS 47 (having an Arg to Pro substitution at position 216 and a Histo Tyr substitution at position 333); RTS 48-2 (having an Ala to Valsubstitution at position 177); and RTS 60-1 (having an Ala to Valsubstitution at position 396).

BRIEF SUMMARY OF THE INVENTION

[0018] The subject invention pertains to materials and methods usefulfor improving crop yields in plants, such as those plants that producecereal crops. In one embodiment, the subject invention provides heatstable AGP enzymes and nucleotide sequences which encode these enzymes.In a preferred embodiment, the heat stable enzymes of the invention canbe used to provide plants having greater tolerance to highertemperatures, thus enhancing the crop yields from these plants. In aparticularly preferred embodiment, the improved plant is a cereal.Cereals to which this invention applies include, for example, maize,wheat, rice, and barley.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows the genomic nucleotide sequence of a wild typeShrunken-2 allele of Zea mays. Introns are indicated by lower caseletters. Base number 1 is the transcription start site.

[0020]FIG. 2 shows a comparison of enzyme activity for wild type andvarious maize AGP large subunit mutants. All reactions were performed induplicate. Numbers given are the average of the duplicates, afterbackground removal. The percentages refer to activity remaining afterheat treatment as compared to activity prior to heat treatment. Thelegend for the figure is as follows:

[0021] “sh2”=wild type sh2 protein;

[0022] “sh2ht”=wild type sh2 protein, following heat treatment;

[0023] “33”=sh2 protein containing the HS 33 mutation (i.e., ahistidine-to-tyrosine amino acid substitution at position 333 in thelarge subunit of maize AGP);

[0024] “33ht”=sh2 protein containing the HS 33 mutation, following heattreatment;

[0025] “177”=sh2 protein containing the mutation rts48-2 (i.e., analanine-to-valine amino acid substitution at position 177 in the largesubunit of maize AGP);

[0026] “177ht”=sh2 protein containing the mutation rts48-2 (i.e., analanine-to-valine amino acid substitution at position 177 in the largesubunit of maize AGP), following heat treatment;

[0027] “396”=sh2 protein containing the mutation rts60-1 (i.e., analanine-to-valine amino acid substitution at position 396 in the largesubunit of maize AGP);

[0028] “396ht”=sh2 protein containing the mutation rts60-1 (i.e., analanine-to-valine amino acid substitution at position 396 in the largesubunit of maize AGP), following heat treatment;

[0029] “7+6”=sh2 protein containing the combination of “177” and “396”mutations;

[0030] “7+6ht”=sh2 protein containing the combination of “177” and “396”mutations, following heat treatment;

[0031] “7+3”=sh2 protein containing the combination of “177” and “HS 33”mutations;

[0032] “7+3ht”=sh2 protein containing the combination of “177” and “HS33” mutations, following heat treatment;

[0033] “6+3”=sh2 protein containing the combination of “396” and “HS 33”mutations;

[0034] “6+3ht”=sh2 protein containing the combination of “396” and “HS33” mutations, following heat treatment.

[0035]FIG. 3 shows a restriction map of Sh2 coding region. Restrictionenzymes shown are those used in isolation of entire coding region and increation of double and triple mutants. Mutations are indicated withasterisks (*)

BRIEF DESCRIPTION OF THE SEQUENCES

[0036] SEQ ID NO. 1 is an amino acid sequence of a region correspondingto amino acids 318 to 350 of the large subunit of AGP in maizecontaining the HS 33 mutation.

[0037] SEQ ID NO. 2 is an amino acid sequence of a region correspondingto amino acids 170 to 189 of the large subunit of AGP in maizecontaining the RTS48-2 mutation.

[0038] SEQ ID NO. 3 is an amino acid sequence of a region correspondingto amino acids 389 to 406 of the large subunit of AGP in maizecontaining the RTS60-1 mutation.

[0039] SEQ ID NO. 4 is the genomic nucleotide sequence of a wild typeShrunken-2 allele of Zea mays.

[0040] SEQ ID NO. 5 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

[0041] SEQ ID NO. 6 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

[0042] SEQ ID NO. 7 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

[0043] SEQ ID NO. 8 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

[0044] SEQ ID NO. 9 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

[0045] SEQ ID NO. 10 is a synthetic oligonucleotide primer that can beused in accordance with the subject invention.

DETAILED DISCLOSURE OF THE INVENTION

[0046] The subject invention concerns novel mutant polynucleotidemolecules, and the polypeptides encoded thereby, that confer increasedheat resistance and yield in plants grown under conditions of heatstress relative to plants expressing wild type genotype. In specificembodiments, the polynucleotide molecules of the subject inventionencode maize endosperm ADP glucose pyrophosphorylase (AGP) and solublestarch synthase (SSS) enzyme activities.

[0047] The mutant enzymes confer increased stability to heat stressconditions during seed and plant development in seeds and plant tissueexpressing the enzymes as compared with wild type enzyme activities. Oneaspect of the subject invention concerns polynucleotides which encodetwo or more amino acid changes in an AGP large subunit as compared tothe wild type sequence of the AGP large subunit polypeptide, wherein theexpressed mutant protein exhibits increased stability. Preferably, thepolypeptide encoded bythe subject polynucleotides, when expressed withthe small subunit, exhibit increased enzymatic activity as compared towild type protein and, preferably, at a level about the same or greaterthan that exhibited by a single amino acid mutation that confersincreased heat stability, such as HS 33. The polynucleotides of theinvention may encode two, three, or more amino acid changes from thewild type sequence. Preferably, a polynucleotide of the inventionencodes a polypeptide having an amino acid substitution at one or moreof the following positions corresponding to the position in the largesubunit of maize AGP: position 177, 333, and 396.

[0048] In one embodiment, a polynucleotide of the present inventionencodes a mutant large subunit of a plant AGP containing a doublemutation: a histidine-to-tyrosine amino acid substitution and analanine-to-valine amino acid substitution in the sequence of thepolypeptide. In an exemplified embodiment, the histidine to tyrosinesubstitution occurs at the amino acid corresponding to residue number333 in the sequence of the large subunit of maize AGP. In oneembodiment, the alanine-to-valine substitution occurs at the amino acidcorresponding to residue number 177 in the sequence of the large subunitof maize AGP. In another embodiment, the alanine-to-valine substitutionoccurs at the amino acid corresponding to residue 396 in the sequence ofthe large subunit of maize AGP.

[0049] In a further embodiment, a polynucleotide of the presentinvention encodes a mutant large subunit of a plant AGP containing twoalanine-to-valine amino acid substitutions within the sequence of thepolypeptide. In an exemplified embodiment, the first alanine-to-valinesubstitution occurs at the amino acid corresponding to residue number177 and the second alanine-to-valine substitution occurs at the aminoacid corresponding to residue number 396 in the sequence of the largesubunit of maize AGP. Enzyme activity associated with mutant proteins ofthe present invention having two mutations are shown in FIG. 2.

[0050] Another embodiment concerns a triple mutant comprising ahistidine to tyrosine substitution at the amino acid corresponding toresidue number 333, an alanine-to-valine substitution at the amino acidcorresponding to residue number 177, and an alanine-to-valinesubstitution at the amino acid corresponding to residue 396 in thesequence of the large subunit of maize AGP.

[0051] The amino acid residue numbers referred to above are based on theaccepted number of the amino acids in this protein (Shaw and Hannah,1992, supra). The position of these substitutions can be readilyidentified by a person skilled in the art. Table 1 below shows thedouble and triple amino acid substitution mutants exemplified herein.TABLE 1 Sh2 Polypeptide Mutant Amino Acid Change HS 7 + 3 Ala to Val atposition 177 and His to Tyr at position 333 HS 6 + 3 Ala to Val atposition 396 and His to Tyr at position 333 HS 7 + 6 Ala to Val atposition 177 and Ala to Val at position 396 HS 7 + 6 + 3 Ala to Val atposition 177 and Ala to Val at position 396 and His to Tyr at position333

[0052] Because of the homology of AGP polypeptides between variousspecies of plants (Smith-White and Preiss [1992] J. Mol. Evol.34:449-464), the ordinarily skilled artisan can readily determine theposition of the mutations in AGP from plants other than maize thatcorrespond to the position of mutations in maize AGP as disclosedherein. Thus, the present invention encompasses polynucleotides thatencode mutant AGP of plants other than maize, including, but not limitedto, wheat, barley, oats, and rice, that confers increased heat stabilitywhen expressed in the plant.

[0053] Single amino acid mutations in AGP that confer heat stability,and methods for producing and selecting for such mutations, aredisclosed in U.S. Pat. No. 6,069,300 and published internationalapplications WO 99/58698 and WO 98/22601. Typically, a plasmidcomprising a polynucleotide coding for the SH2 subunit of maize AGP wasmutagenized, placed into mutant E. coli glg C⁻¹ cells expressing the BT2subunit, and the cells grown at 42° C. to select for mutants that couldproduce glycogen at that temperature. Several mutants, termed heatstable (HS) mutants, were isolated. Crude extracts of these mutants wereprepared and the heat stability of the resulting AGP was monitored. Thesingle amino acid substitution mutants retained between 8-59% of theiractivity after incubation at 60° C. for five minutes. In addition, totalenzymatic activity of the mutant maize endosperm AGP before heattreatment was elevated about two- to three-fold in several of themutants.

[0054] Multiple heat stability conferring mutations can easily becombined within one subunit. For example, different unique restrictionsites that divide the coding regions of Sh2 into three distinctfragments can be used. Where appropriate, mutation combinations can begenerated by subcloning the corresponding fragment containing the addedmutation. If two mutations are in close proximity, then site-directedmutagenesis can be used to engineer such combinations. One method forsite specific mutations involves PCR, mutagenic primer, and the useofDpnI restriction endonuclease. Primers can be constructed to containthe mutation in the 5′ end, and used to PCR amplify using theproofreading polymerase Vent. Amplified DNA can then be digested withDpnI. Parental DNA isolated from E. coli is methylated and hencesusceptible to DpnI. Digested DNA is size fractionated by gelelectrophoresis, ligated, and cloned into the expression vectors.Mutations are confirmed by sequence analysis and transformed into theAC70R1-504 strain carrying the wild type small subunit. Combinatorialmutants can then be analyzed.

[0055] The subject invention also concerns the mutant polypeptides,encoded by the subject polynucleotides, having the amino acidsubstitutions described herein. In a preferred embodiment, the mutantpolypeptides are from maize.

[0056] The subject invention also concerns heat stable mutants of AGP ofthe present invention combined with heat stable mutations in the smallsubunit of the enzyme. Mutations in the small subunit of AGP that conferheat stability to the enzyme can also be readily prepared and identifiedusing the methods described in U.S. Pat. No. 6,069,300 and publishedinternational applications WO 99/58698 and WO 98/22601. Heat stablemutants of the small subunit can be co-expressed with the mutants of thepresent invention to further enhance the stability of an AGP enzyme.

[0057] Plants and plant tissue bred to contain or transformed with themutant polynucleotides of the invention, and expressing the polypeptidesencoded by the polynucleotides, are also contemplated by the presentinvention. Plants and plant tissue expressing the mutant polynucleotidesproduce tissues that have, for example, lower heat-induced loss inweight or yield when subjected to heat stress during development. Plantswithin the scope of the present invention include monocotyledonousplants, such as rice, wheat, barley, oats, sorghum, maize, lilies, andmillet, and dicotyledonous plants, such as peas, alfalfa, chickpea,chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato,sweet potato, radish, cabbage, rape, apple trees, and lettuce. In aparticularly preferred embodiment, the plant is a cereal. Cereals towhich this invention applies include, for example, maize, wheat, rice,barley, oats, rye, and millet.

[0058] The subject invention also concerns methods for producing andidentifying polynucleotides and polypeptides contemplated within thescope of the invention. In one embodiment, gene mutation, followed byselection using a bacterial expression system, can be used to isolatepolynucleotide molecules that encode plant AGP subunits that possessmutations that can alleviate heat-induced loss in starch synthesis inplants. Individual amino acid substitutions can be combined into onesubunit as described herein.

[0059] The subject invention further concerns plants and plant tissuethat comprise a polynucleotide of the present invention that encodes amutant polypeptide of the invention. In a preferred embodiment, theplant or plant tissue has an AGP mutant gene of the inventionincorporated into its genome. Other alleles that confer advantageousphenotypes can also be incorporated into a plant genome. In a preferredembodiment, the plant is a cereal plant. More preferably, the plant isZea mays. Plants having an AGP mutant gene can be grown from seeds thatcomprise a mutant gene in their genome. In addition, techniques fortransforming plants with a gene, such as Agrobacterium infection,biolistic methods, etc., are known in the art.

[0060] Because of the degeneracy of the genetic code, a variety ofdifferent polynucleotide sequences can encode each of the variant AGPpolypeptides disclosed herein. In addition, it is well within the skillof a person trained in the art to create alternative polynucleotidesequences encoding the same, or essentially the same, polypeptides ofthe subject invention. These variant or alternative polynucleotidesequences are within the scope of the subject invention. As used herein,references to “essentially the same” sequence refers to sequences whichencode amino acid substitutions, deletions, additions, or insertionswhich do not materially alter the functional activity of the polypeptideencoded by the AGP mutant polynucleotide described herein.

[0061] As used herein, the terms “nucleic acid” and “polynucleotidesequence” refer to a deoxyribonucleotide or ribonucleotide polymer ineither single- or double-stranded form, and unless otherwise limited,would encompass known analogs of natural nucleotides that can functionin a similar manner as naturally-occurring nucleotides. Thepolynucleotide sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The polynucleotide sequences include both full-length sequencesas well as shorter sequences derived from the full-length sequences. Itis understood that a particular polynucleotide sequence includes thedegenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell. Allelicvariations of the exemplified sequences also come within the scope ofthe subject invention. The polynucleotide sequences falling within thescope of the subject invention further include sequences whichspecifically hybridize with the exemplified sequences. Thepolynucleotide includes both the sense and antisense strands as eitherindividual strands or in the duplex.

[0062] Substitution of amino acids other than those specificallyexemplified in the mutants disclosed herein are also contemplated withinthe scope of the present invention. Amino acids can be placed in thefollowing classes: non-polar, uncharged polar, basic, and acidic.Conservative substitutions whereby a mutant AGP polypeptide having anamino acid of one class is replaced with another amino acid of the sameclass fall within the scope of the subject invention so long as themutant AGP polypeptide having the substitution still retains increasedheat stability relative to a wild type polypeptide. Table 2 belowprovides a listing of examples of amino acids belonging to each class.TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

[0063] For example, substitution of the tyrosine at position 333 in theHS 33, HS 7+3, HS 6+3, and HS 7+6+3 mutants with other amino acids, suchas glycine, serine, threonine, cysteine, asparagine, and glutamine, areencompassed within the scope of the invention. Also specificallycontemplated within the scope of the invention is substitution of eithera phenylalanine or a methionine at position 333 in the AGP largesubunit. Thus, a combination of phenylalanine or methionine at position333 with either a valine at position 177 or a valine at position 396, orboth, is specifically contemplated by the present invention. Similarly,substitution of the valine at positions 177 and 396 in the RTS 48-2, RTS60-1, HS 7+3, HS 6+3, HS 7+6, and HS 7+6+3 mutants with other aminoacids such as leucine, isoleucine, proline, methionine, phenylalanine,and tryptophan, is within the scope of the invention. Amino acidsubstitutions at positions other than the site of the heat stablemutation are also contemplated within the scope of the invention so longas the polypeptide retains or confers increased heat stability relativeto wild type polypeptides.

[0064] Polynucleotides and proteins of the subject invention can also bedefined in terms of more particular identity and/or similarity rangeswith those exemplified herein. The sequence identity will typically begreater than 60%, preferably greater than 75%, more preferably greaterthan 80%, even more preferably greater than 90%, and can be greater than95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (Karlin and Altschul [1990]Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin andAltschul (Karlin and Altschul [1993] Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al. (Altschul et al. [1990] J Mol. Biol.215:402-410). BLAST searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain sequences with the desired percentsequence identity. To obtain gapped alignments for comparison purposes,Gapped BLAST can be used as described in Altschul et al. (Altschul etal. [1997] Nucl. Acids Res. 25:3389-3402). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) can be used. See NCBI/NIH website.

[0065] The subject invention also concerns polynucleotides which encodefragments of the full length mutant polypeptide, so long as thosefragments retain substantially the same functional activity as fulllength polypeptide. The fragments of mutant AGP polypeptide encoded bythese polynucleotides are also within the scope of the presentinvention. Fragments of the full length sequence can be prepared usingstandard techniques known in the art.

[0066] The subject invention also contemplates those polynucleotidemolecules encoding starch biosynthesis enzymes having sequences whichare sufficiently homologous with the wild type sequence so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis, T., E. F. Fritsch, J. Sambrook [1982]Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). As used herein, “stringent” conditions forhybridization refers to conditions wherein hybridization is typicallycarried out overnight at 20-25° C. below the melting temperature (Tm) ofthe DNA hybrid in 6× SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/mldenatured DNA. The melting temperature is described by the followingformula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, andF. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K.Moldave [eds.] Academic Press, New York 100:266-285):

[0067] Tm=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.61(%formamide)-600/length of duplex in base pairs.

[0068] Washes are typically carried out as follows:

[0069] (1) Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS(low stringency wash).

[0070] (2) Once at Tm-20° C. for 15 minutes in 0.2× SSPE, 0.1% SDS(moderate stringency wash).

[0071] The polynucleotide molecules of the subject invention can be usedto transform plants to express the mutant heat stable enzyme in thoseplants. In addition, the polynucleotides of the subject invention can beused to express the recombinant variant enzyme. They can also be used asa probe to detect related enzymes. The polynucleotides can also be usedas DNA sizing standards.

[0072] The polynucleotide molecules of the subject invention alsoinclude those polynucleotides that encode starch biosynthesis enzymes,such as AGP enzymes, that contain mutations that can confer increasedseed weight, in addition to enhanced heat stability, to a plantexpressing these mutants. The combination of a heat stabilizingmutation, such as, for example, Sh2-HS 7+6 or Sh2-HS 7+3, with amutation conferring increased seed weight, e.g., Rev6, in apolynucleotide that encodes the large subunit of maize AGP isspecifically contemplated in the present invention. U.S. Pat. Nos.5,589,618 and 5,650,557 disclose polynucleotides (e.g., Rev6) thatencode mutations in the large subunit of AGP that confer increased seedweight in plants that express the mutant polypeptide.

[0073] Mutations in the AGP subunits that confer heat stability can becombined according to the subject invention with phosphate insensitivemutants of maize, such as the Rev6 mutation, to enhance the stability ofthe Rev6 encoded large subunit.

[0074] It is expected that enzymic activity of SSS will be impaired athigher temperatures as observed with AGP. Thus, mutagenized forms of SSScan be expressed under increased thermal conditions (42° C.), to isolateheat stable variants in accordance with the methods described herein.These heat stable mutagenized forms of SSS are further aspects of thesubject invention.

[0075] The subject invention also concerns methods for increasing yieldcharacteristics of plants under conditions of heat stress byincorporating a polynucleotide of the present invention that comprises amutation in a starch biosynthesis enzyme that confers increasedstability or resistance to heat stress conditions and a mutation thatconfers increased yield characteristics on the plant. Increased yieldcharacteristics include, for example, increased seed number, increasedseed weight, increased plant biomass, and increased Harvest Index.

[0076] All patents, patent applications, provisional applications, andpublications referred to or cited herein are hereby incorporated byreference in their entirety to the extent they are not inconsistent withthe explicit teachings of this specification.

[0077] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Testing of Maize Endosperm ADP-glucose Pyrophosphorylaseshaving Multiple Amino Acid Mutations

[0078] Expression of Maize Endosperm ADP-glucose Pyrophosphorylases.10-ml aliquots of Luria broth (75 g/mL of spectinomycin and 50 g/mL ofkanamycin) were inoculated from glycerol stocks of AC70R1-504 E. colicells expressing either maize endosperm or potato tuber ADP-glucosepyrophosphorylase, and grown overnight at 37C with shaking at 220 rpm.These cultures were used to inoculate 250 mL of Luria broth (75 g/mL ofspectinomycin and 50 g/mL of kanamycin). Cultures were grown to anOD₆₀₀=0.55 at 37C with shaking at 220 rpm. Cultures were induced with0.2 mM isopropyl -D-thiogalactoside and 0.02 mg/mL nalidixic acid for 7hrs at room temperature with shaking at 220 rpm. Cells were harvested at3500 rpm for 10 min at 4C. Cell pellets were resuspended in 800L ofextraction buffer: 50 mM HEPES, pH 7.5,5 mM MgCl₂, 5 mM EDTA, 20%sucrose, and 30% ammonium sulfate. DTT (1 mM), 50 g/ml lysozyme, 1 g/mLpepstatin, 1 g/mL leupeptin, 1 g/mL antipain, 10 g/mL chymostatin, 1 mMphenylmethylsulfonyl fluoride, and 1 mM benzamidine were added toextraction buffer just before use. Lysates were sonicated three timesfor three seconds with incubation on ice between sonications. Sampleswere centrifuged for 1 min at 13,000 rpm at 4C. Supernatants wereremoved and aliquoted for assays.

[0079] Combination of Individual Mutations. A subcloning strategy wasdesigned to study the effects of the mutations in combination with HS33, and with each other. To combine the reversion mutations of RTS 48-2and RTS 60-1, the plasmids containing each reversion mutation (thetemperature sensitive parental mutations were removed prior to combiningmutations) were digested with Eco RV and a 339 bp fragment of RTS 48-2was exchanged for the corresponding fragment of RTS 60-1 (FIG. 3). Theresulting plasmid was designated Sh2-HS 7+6. A similar strategy was usedto combine the reversion mutation of RTS 48-2 with the mutationidentified in HS 33. Plasmids containing the mutations were digestedwith Eco RV and a 339 bp fragment of RTS 48-2 was exchanged for thecorresponding fragment of HS 33. The resulting plasmid was designatedSh2-HS 7+3. To combine the reversion mutation of RTS 60-1 with themutation identified in HS 33, plasmids containing the mutations weredigested with Mun I/Kpn I and a 390 bp fragment of RTS 60-1 wasexchanged for the corresponding fragment of HS 33 (FIG. 3). Theresulting plasmid was designated Sh2-HS 6+3. In order to combine thereversion mutations of RTS 60-1 and RTS 48-2 with the mutationidentified in HS 33, plasmid Sh2-HS 6+3 and a plasmid containing thereversion mutation RTS 48-2 were digested with Eco RV and a 339 bpfragment of RTS 48-2 was exchanged for the corresponding fragment ofSh2-HS 6+3. The resulting plasmid was designated Sh2-HS 7+6+3.

[0080] Final sequencing of all plasmids was performed using six primersto cover the entire Sh2 coding region in both directions. Primers usedare as follows:

[0081] LHBB1 (5′→3′): 5′-CGACTCACTATAGGGAGACC-3′ (SEQ ID NO. 5);

[0082] LH27 (5′→3′): 5′-CCCTATGAGTAACTG-3′ (SEQ ID NO. 6);

[0083] LH9 (5′→3′): 5′-TATACTCAATTACAT-3′ (SEQ ID NO. 7);

[0084] LHBB2 (3′→5′): 5′-GTGCCACCTGACGTCTAAG-3′ (SEQ ID NO. 8);

[0085] LH2135 (3′→5′): 5′-CAGAGCTGACACGTG-3′ (SEQ ID NO. 9);

[0086] LH32 (3′→5′): 5′-AAGCTGATCGCCACTC-3′ (SEQ ID NO. 10).

[0087] Heat Treatment of ADP-glucose Pyrophosphorylase. Wild type (sh2)and mutant ADP-glucose pyrophosphorylase containing a single amino acidmutation (HS 33, RTS 48-2, RTS 60-1) and multiple mutation (HS 7+3,i.e., RTS 48-2 plus HS 33; HS 6+3, i.e., RTS 60-1 plus HS 33; and HS7+6, i.e., RTS 48-2 plus RTS 60-1) amino acid changes were tested forenzyme activity before and after heat treatment. Heat treatmentconsisted of incubation of the test protein at 60° C. for 5 minutes.

[0088] The percentage of activity remaining after heat treatment at 60°C. for 5 min is presented in Table 3. Genotypes in the data set are Sh2wild type, HS 33, RTS 60-1 (reversion mutation only), RTS 48-2(reversion mutation only), Sh2-HS 7+6, Sh2-HS 6+3, Sh2-HS 7+3, andSh2-HS 7+6+3. TABLE 3 Percent Activity Remaining After Heat TreatmentEnzyme % Activity SEM^(a) N^(b) Sh2 wt 32 11 3 HS 33 69  7 7 RTS 60-1 61 13* 2 RTS 48-2 64  6 3 Sh2-HS 7 + 6 77  21* 2 Sh2-HS 6 + 3 69  9 3Sh2-HS 7 + 3 83  8 3 Sh2-HS 7 + 6 + 3 72 11 3

[0089] Activity before heat treatment for Sh2 wild type, HS 33, RTS60-1, RTS 48-2, Sh2-HS 7+6, Sh2-HS 6+3, Sh2-HS 7+3, and Sh2-HS 7+6+3 isshown in Table 4. HS 33 has 2.1 fold more activity than does Sh2 wildtype. Both RTS 48-2 and RTS 60-1 show a 1.4 fold increase in activity.Their double mutant contains a 1.9 fold increase in activity. While thecombination of the two mutants increases activity, the double mutantdoes not experience synergistic effects. The mutation ofRTS 60-1 whencombined with that of HS 33 experiences an additive effect, raisingactivity 3.4 fold compared to Sh2 wild type. The mutation of RTS 48-2 incombination with that of HS 33 exhibits a slightly smaller increase to2.9 fold. Interestingly, the triple mutant shows a slightly greaterincrease than either second-site reversion mutation alone, but less thanthe double mutant between second-site revertants. TABLE 4 Fold Increasein Activity Enzyme Fold increase Range N^(b) Sh2 wt n/a n/a n/a HS 332.1 0.2^(a) 3 RTS 60-1 1.4 0 1 RTS 48-2 1.4 0 1 Sh2-HS 7 + 6 1.9 0.2 2Sh2-HS 6 + 3 3.4 0 1 Sh2-HS 7 + 3 2.9 0.1 2 Sh2-HS 7 + 6 + 3 1.8 0.1 2

[0090] ADP-glucose Pyrophosphorylase Assays. To obtain quantitative datafor the mutants described above, activity was measured with thesynthesis (forward) assay that measures incorporation Of[¹⁴C]glucose-1-P into the sugar nucleotide ADP-glucose. Assays wereperformed on crude enzyme extracts prepared as described below.

[0091] The ADP-glucose synthesis reaction measures incorporation of[¹⁴C]glucose-1-P into ADP-Glucose. The reaction mixture contained 80 mMHEPES, pH 7.5m, 1 mM glucose-1-P, 4 MM MgCl₂, 0.5 mg mL⁻¹; bovine serumalbumin, 10 mM 3-PGA, and 15,000 cpm of [¹⁴C]glucose-1-P. Reactionvolume was 50 mL. Assays were initiated by addition of 1.5 mM ATP.Reaction was incubated for 30 min at 37° C. and terminated by boilingfor 2 min. Unincorporated glucose-1-P was cleaved by addition of 0.3 Uof bacterial alkaline phosphatase (Worthington Biochemical Corporation,Lakewood, N.J.) and incubation for 2.5 h at 37° C. A 20 mL aliquot ofthe reaction mixture was spotted on DEAE paper, washed with distilledwater three times, dried, and quantified in a liquid scintillationcounter.

[0092] Additional results for single and double mutants are shown inFIG. 2. For the combination mutant Sh2-HS 7+6 (RTS 48-2 plus RTS 60-1)and the combination mutant Sh2-HS 6+3 (RTS 60-1 plus HS 33), the numbersgiven are the average of data from 3 dilutions of the enzyme (induplicate), multiplied by their dilution factor, minus background. Forthe combination Sh2-HS 7+3 (RTS 48-2 plus HS 33) mutant, the numbersgiven are the average of 2 dilutions of the enzyme (in duplicate),multiplied by their dilution factor, minus background. Graphicrepresentation of the numbers was performed using Microsoft Excel.

EXAMPLE 2 Combination of Heat Stability Mutations with Rev6

[0093] According to the subject invention, the heat stable mutations canbe combined with a mutation associated with increased seed weight, suchas, for example, the Rev6 mutation. The goal is to maintain the desiredphosphate insensitivity characteristic of Rev6 while enhancing itsstability. Mutants comprising heat stable mutations combined with Rev6mutation can be constructed and confirmed as described herein. These“combination” mutants can be transformed into AC70R1-504 carrying thewild type small subunit. Increased heat stability can be easilyidentified by a positive glycogen staining on a low glucose media. Rev6does not stain when grown on this media. Initially all mutantcombinations can be screened enzymatically for maintenance of phosphateinsensitivity, and only combinations that maintain phosphateinsensitivity are further analyzed.

[0094] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are included within the spirit and purview ofthis application and the scope of the appended claims.

1 10 1 33 PRT HS 33 Mutant of Zea mays 1 Leu His Asp Phe Gly Ser Glu IleLeu Pro Arg Ala Val Leu Asp Tyr 1 5 10 15 Ser Val Gln Ala Cys Ile PheThr Gly Tyr Trp Glu Asp Val Gly Thr 20 25 30 Ile 2 20 PRT RTS48-2 Mutantof Zea mays 2 Thr Gln Met Pro Glu Glu Pro Val Gly Trp Phe Gln Gly ThrAla Asp 1 5 10 15 Ser Ile Arg Lys 20 3 18 PRT RTS60-1 Mutant of Zea mays3 Asp Lys Cys Lys Met Lys Tyr Val Phe Ile Ser Asp Gly Cys Leu Leu 1 5 1015 Arg Glu 4 7739 DNA Wild-type Shrunken-2 allele of Zea mays 4taagaggggt gcacctagca tagatttttt gggctccctg gcctctcctt tcttccgcct 60gaaaacaacc tacatggata catctgcaac cagagggagt atctgatgct ttttcctggg 120cagggagagc tatgagacgt atgtcctcaa agccactttg cattgtgtga aaccaatatc 180gatctttgtt acttcatcat gcatgaacat ttgtggaaac tactagctta caagcattag 240tgacagctca gaaaaaagtt atctctgaaa ggtttcatgt gtaccgtggg aaatgagaaa 300tgttgccaac tcaaacacct tcaatatgtt gtttgcaggc aaactcttct ggaagaaagg 360tgtctaaaac tatgaacggg ttacagaaag gtataaacca cggctgtgca ttttggaagt 420atcatctata gatgtctgtt gaggggaaag ccgtacgcca acgttattta ctcagaaaca 480gcttcaacac acagttgtct gctttatgat ggcatctcca cccaggcacc caccatcacc 540tattcaccta tctctcgtgc ctgtttattt tcttgccctt tctgatcata aaaaatcatt 600aagagtttgc aaacatgcat aggcatatca atatgctcat ttattaattt gctagcagat 660catcttccta ctctttactt tatttattgt ttgaaaaata tgtcctgcac ctagggagct 720cgtatacagt accaatgcat cttcattaaa tgtgaatttc agaaaggaag taggaaccta 780tgagagtatt tttcaaaatt aattagcggc ttctattatg tttatagcaa aggccaaggg 840caaaatcgga acactaatga tggttggttg catgagtctg tcgattactt gcaagaaatg 900tgaacctttg tttctgtgcg tgggcataaa acaaacagct tctagcctct tttacggtac 960ttgcacttgc aagaaatgtg aactcctttt catttctgta tgtggacata atgccaaagc 1020atccaggctt tttcatggtt gttgatgtct ttacacagtt catctccacc agtatgccct 1080cctcatactc tatataaaca catcaacagc atcgcaatta gccacaagat cacttcggga 1140ggcaagtgtg atttcgacct tgcagccacc tttttttgtt ctgttgtaag tatactttcc 1200cttaccatct ttatctgtta gtttaatttg taattgggaa gtattagtgg aaagaggatg 1260agatgctatc atctatgtac tctgcaaatg catctgacgt tatatgggct gcttcatata 1320atttgaattg ctccattctt gccgacaata tattgcaagg tatatgccta gttccatcaa 1380aagttctgtt ttttcattct aaaagcattt tagtggcacg caattttgtc catgagggaa 1440aggaaatctg ttttggttac tttgcttgag gtgcattctt catatgtcca gttttatgga 1500agtaataaac ttcagtttgg tcataagatg tcatattaaa gggcaaacat atattcaatg 1560ttcaattcat cgtaaatgtt ccctttttgt aaaagattgc atactcattt atttgagttg 1620caggtgtatc tagtagttgg aggagatatg cagtttgcac ttgcattgga cacgaactca 1680ggtcctcacc agataagatc ttgtgagggt gatgggattg acaggttgga aaaattaagt 1740attgggggca gaaagcagga gaaagctttg agaaataggt gctttggtgg tagagttgct 1800gcaactacac aatgtattct tacctcagat gcttgtcctg aaactcttgt aagtatccac 1860ctcaattatt actcttacat gttggtttac tttacgtttg tcttttcaag ggaaatttac 1920tgtatttttt gtgttttgtg ggagttctat acttctgttg gactggttat tgtaaagatt 1980tgttcaaata gggtcatcta ataattgttt gaaatctggg aactgtggtt tcactgcgtt 2040caggaaaaag tgaattattg gttactgcat gaataactta tggaaataga ccttagagtt 2100gctgcatgat tatcacaaat cattgctacg atatcttata atagttcttt cgacctcgca 2160ttacatatat aactgcaact cctagttgcg ttcaaaaaaa aaaatgcaac tcttagaacg 2220ctcaccagtg taatctttcc tgaattgtta tttaatggca tgtatgcact acttgtatac 2280ttatctagga ttaagtaatc taactctagg ccccatattt gcagcattct caaacacagt 2340cctctaggaa aaattatgct gatgcaaacc gtgtatctgc tatcattttg ggcggaggca 2400ctggatctca gctctttcct ctgacaagca caagagctac gcctgctgta agggataaca 2460ctgaacatcc aacgttgatt actctattat agtattatac agactgtact tttcgaattt 2520atcttagttt tctacaatat ttagtggatt cttctcattt tcaagataca caattgatcc 2580ataatcgaag tggtatgtaa gacagtgagt taaaagatta tattttttgg gagacttcca 2640gtcaaatttt cttagaagtt tttttggtcc agatgttcat aaagtcgccg ctttcatact 2700ttttttaatt ttttaattgg tgcactatta ggtacctgtt ggaggatgtt acaggcttat 2760tgatatccct atgagtaact gcttcaacag tggtataaat aagatatttg tgatgagtca 2820gttcaattct acttcgctta accgccatat tcatcgtaca taccttgaag gcgggatcaa 2880ctttgctgat ggatctgtac aggtgattta cctcatcttg ttgatgtgta atactgtaat 2940taggagtaga tttgtgtgga gagaataata aacagatgcc gagattcttt tctaaaagtc 3000tagatccaaa ggcattgtgg ttcaaaacac tatggacttc taccatttat gtcattactt 3060tgccttaatg ttccattgaa tggggcaaat tattgattct acaagtgttt aattaaaaac 3120taattgttca tcctgcaggt attagcggct acacaaatgc ctgaagagcc agctggatgg 3180ttccagggta cagcagactc tatcagaaaa tttatctggg tactcgaggt agttgatatt 3240ttctcgttta tgaatgtcca ttcactcatt cctgtagcat tgtttctttg taattttgag 3300ttctcctgta tttctttagg attattacag tcacaaatcc attgacaaca ttgtaatctt 3360gagtggcgat cagctttatc ggatgaatta catggaactt gtgcaggtat ggtgttctct 3420tgttcctcat gtttcacgta atgtcctgat tttggattaa ccaactactt ttggcatgca 3480ttatttccag aaacatgtcg aggacgatgc tgatatcact atatcatgtg ctcctgttga 3540tgagaggtaa tcagttgttt atatcatcct aatatgaata tgtcatcttg ttatccaaca 3600caggatgcat atggtctaat ctgctttcct tttttttccc ttcggaagcc gagcttctaa 3660aaatgggcta gtgaagattg atcatactgg acgtgtactt caattctttg aaaaaccaaa 3720gggtgctgat ttgaattcta tggttagaaa ttccttgtgt aatccaattc ttttgttttc 3780ctttctttct tgagatgaac ccctctttta gttatttcca tggataacct gtacttgact 3840tattcagaaa tgattttcta ttttgctgta gaatctgaca ctaaagctaa tagcactgat 3900gttgcagaga gttgagacca acttcctgag ctatgctata gatgatgcac agaaatatcc 3960ataccttgca tcaatgggca tttatgtctt caagaaagat gcacttttag accttctcaa 4020gtaatcactt tcctgtgact tatttctatc caactcctag tttaccttct aacagtgtca 4080attcttaggt caaaatatac tcaattacat gactttggat ctgaaatcct cccaagagct 4140gtactagatc atagtgtgca ggtaagtctg atctgtctgg agtatgtgtt ctgtaaactg 4200taaattcttc atgtcaaaaa gttgtttttg tttccagttt ccactaccaa tgcacgattt 4260atgtattttc gcttccatgc atcatacata ctaacaatac attttacgta ttgtgttagg 4320catgcatttt tacgggctat tgggaggatg ttggaacaat caaatcattc tttgatgcaa 4380acttggccct cactgagcag gtactctgtc atgtattctg tactgcatat atattacctg 4440gaattcaatg catagaatgt gttagaccat cttagttcca tcctgttttc ttcaattagc 4500ttatcattta atagttgttg gctagaattt aaacacaaat ttacctaata tgtttctctc 4560ttcagccttc caagtttgat ttttacgatc caaaaacacc tttcttcact gcaccccgat 4620gcttgcctcc gacgcaattg gacaagtgca aggtatatgt cttactgagc acaattgtta 4680cctgagcaag attttgtgta cttgacttgt tctcctccac agatgaaata tgcatttatc 4740tcagatggtt gcttactgag agaatgcaac atcgagcatt ctgtgattgg agtctgctca 4800cgtgtcagct ctggatgtga actcaaggta catactctgc caatgtatct actcttgagt 4860ataccatttc aacaccaagc atcaccaaat cacacagaac aatagcaaca aagcctttta 4920gttccaagca atttagggta gcctagagtt gaaatctaac aaaacaaaag tcaaagctct 4980atcacgtgga tagttgtttt ccatgcactc ttatttaagc taattttttg ggtatactac 5040atccatttaa ttattgtttt attgcttctt ccctttgcct ttcccccatt actatcgcgt 5100cttaagatca tactacgcac tagtgtcttt agaggtctct ggtggacatg ttcaaaccat 5160ctcaatcggt gttggacaag tttttcttga atttgtgcta cacctaacct atcacgtatg 5220tcatcgtttc aaactcgatc cttcctgtat catcataaat ccaatgcaac atacgcattt 5280atgcaacatt tatctgttga acatgtcatc tttttgtagg ttaacattat gcaccataca 5340atgtagcatg tctaatcatc atcctataaa atttacattt tagcttatgt ggtatcctct 5400tgccacttag aacaccatat gcttgatgcc atttcatcca ccctgctttg attctatggc 5460taacatcttc attaatatcc tcgcctctct gtatcattgg tcctaaatat ggaaatacat 5520tctttctggg cactacttga ccttccaaac taacgtctcc tttgctcctt tcttgtgtgt 5580agtagtaccg aagtcacatc tcatatattc ggttttagtt ctactaagtc ccgggttcga 5640tccccctcag gggtgaattt cgggcttggt aaaaaaaatc ccctcgctgt gtcccgcccg 5700ctctcgggga tcgatatcct gcgcgccacc ctccggctgg gcattgcaga gtgagcagtt 5760gatcggctcg ttagtgatgg ggagcggggt tcaagggttt tctcggccgg gaccatgttt 5820cggtctctta atataatgcc gggagggcag tctttccctc cccggtcgag ttttagttct 5880accgagtcta aaacctttgg actctagagt cccctgtcac aactcacaac tctagttttc 5940tatttacttc tacctagcgt ttattaatga tcactatatc gtctgtaaaa agcatacacc 6000aatgtaatcc ccttgtatgt cccttgtaat attatccatc acaagaaaaa aaggtaaggc 6060tcaaagttga cttttgatat agtcctattc taatcgagaa gtcatctgta tcttcgtctc 6120ttgttcgaac actagtcaca aaattttttg tacatgttct taatgagtcc aacgtaatat 6180tccttgatat tttgtcataa gccctcatca agtcaatgaa aatcacgtgt aggtccttca 6240tttgttcctt atactgctcc atcacttgtc tcattaagaa aatctctctc atagttaacc 6300ttttggcatg aaacaaaatc acacagaagt tgtttccttt ttttaagatc ccacacaaaa 6360gaggtttgat ctaaggaatc tggatccctg acaggtttat caaaatcctt tgtgtttttc 6420ttaaaactga atattcctcc agcttctagt attgatgtaa tattcaatct gtttagcaag 6480tgaacacctt ggttcttgtt gttactgtac cccccccccc cccccccccc cgaggcccag 6540attaccacga catgaataca agaatattga acccagatct agagtttgtt tgtactgttg 6600aaaatcggtg acaattcatt ttgttattgc gctttctgat aacgacagga ctccgtgatg 6660atgggagcgg acacctatga aactgaagaa gaagcttcaa agctactgtt agctgggaag 6720gtcccagttg gaataggaag gaacacaaag ataaggtgag tatggatgtg gaaccaccgg 6780ttagttccca aaaatatcac tcactgatac ctgatggtat cctctgatta ttttcaggaa 6840ctgtatcatt gacatgaatg ctaggattgg gaagaacgtg gtgatcacaa acagtaaggt 6900gagcgagcgc acctacatgg gtgcagaatc ttgtgtgctc atctatccta attcggtaat 6960tcctatccag cgctagtctt gtgaccatgg ggcatgggtt cgactctgtg acagggcatc 7020caagaggctg atcacccgga agaagggtac tacataaggt ctggaatcgt ggtgatcttg 7080aagaatgcaa ccatcaacga tgggtctgtc atatagatcg gctgcgtgtg cgtctacaaa 7140acaagaacct acaatggtat tgcatcgatg gatcgtgtaa ccttggtatg gtaagagccg 7200cttgacagaa agtcgagcgt tcgggcaaga tgcgtagtct ggcatgctgt tccttgacca 7260tttgtgctgc tagtatgtac tgttataagc tgccctagaa gttgcagcaa acctttttat 7320gaacctttgt atttccatta cctgctttgg atcaactata tctgtcatcc tatatattac 7380taaattttta cgtgtttttc taattcggtg ctgcttttgg gatctggctt cgatgaccgc 7440tcgaccctgg gccattggtt cagctctgtt ccttagagca actccaagga gtcctaaatt 7500ttgtattaga tacgaaggac ttcagccgtg tatgtcgtcc tcaccaaacg ctctttttgc 7560atagtgcagg ggttgtagac ttgtagccct tgtttaaaga ggaatttgaa tatcaaatta 7620taagtattaa atatatattt aattaggtta acaaatttgg ctcgttttta gtctttattt 7680atgtaattag ttttaaaaat agacctatat ttcaatacga aatatcatta acatcgata 7739 520 DNA Artificial sequence Synthetic oligonucleotide primer 5 cgactcactatagggagacc 20 6 15 DNA Artificial Sequence Synthetic oligonucleotideprimer 6 ccctatgagt aactg 15 7 15 DNA Artificial Sequence Syntheticoligonucleotide primer 7 tatactcaat tacat 15 8 19 DNA ArtificialSequence Synthetic oligonucleotide primer 8 gtgccacctg acgtctaag 19 9 15DNA Artificial Sequence Synthetic oligonucleotide primer 9 cagagctgacacgtg 15 10 16 DNA Artificial Sequence Synthetic oligonucleotide primer10 aagctgatcg ccactc 16

We claim:
 1. A polynucleotide encoding a mutant large subunit of a plantADP-glucose pyrophosphorylase polypeptide, or a biologically-activefragment of said mutant polypeptide, wherein said mutant polypeptidecomprises amino acid mutations at two or more sites in the amino acidsequence of said polypeptide and wherein when said mutant polypeptide isexpressed with the small subunit of ADP-glucose pyrophosphorylase toform a mutant ADP-glucose pyrophosphorylase enzyme, said mutant enzyme,or a biologically-active fragment of said mutant enzyme, exhibitsincreased heat stability relative to wild type ADP-glucosepyrophosphorylase enzyme.
 2. The polynucleotide according to claim 1,wherein said mutant enzyme exhibits enzymatic activity substantially thesame or greater than that exhibited by an ADP-glucose pyrophosphorylaseenzyme having only a single amino acid substitution of a histidine totyrosine at position 333 in the amino acid sequence of the wild typelarge subunit of maize.
 3. The polynucleotide according to claim 1,wherein said mutant polypeptide encoded by said polynucleotide comprisesa first amino acid mutation wherein the histidine amino acidcorresponding to position 333 in the amino acid sequence of the wildtype large subunit of ADP-glucose pyrophosphorylase polypeptide of maizeis replaced by an amino acid that confers said increased heat stabilityon said mutant enzyme.
 4. The polynucleotide according to claim 3,wherein the amino acid that replaces histidine at position number 333 isselected from the group consisting of tyrosine, phenylalanine,methionine, glycine, serine, threonine, cysteine, asparagine, andglutamine.
 5. The polynucleotide according to claim 3, wherein the aminoacid that replaces histidine at position number 333 is tyrosine.
 6. Thepolynucleotide according to claim 3, wherein the amino acid thatreplaces histidine at position number 333 is phenylalanine.
 7. Thepolynucleotide according to claim 3, wherein the amino acid thatreplaces histidine at position number 333 is methionine.
 8. Thepolynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises a first amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 9. Thepolynucleotide according to claim 8, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 10. Thepolynucleotide according to claim 8, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 11. Thepolynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises a first amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme
 12. Thepolynucleotide according to claim 11, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 13. Thepolynucleotide according to claim 3, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 14. Thepolynucleotide according to claim 13, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 15. Thepolynucleotide according to claim 13, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 16. Thepolynucleotide according to claim 4, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 17. Thepolynucleotide according to claim 16, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 18. Thepolynucleotide according to claim 16, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 19. Thepolynucleotide according to claim 5, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 20. Thepolynucleotide according to claim 19, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 21. Thepolynucleotide according to claim 19, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 22. Thepolynucleotide according to claim 6, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 23. Thepolynucleotide according to claim 22, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 24. Thepolynucleotide according to claim 22, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 25. Thepolynucleotide according to claim 7, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 177 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 26. Thepolynucleotide according to claim 25, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 27. Thepolynucleotide according to claim 25, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 28. Thepolynucleotide according to claim 3, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide ofmaize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 29. Thepolynucleotide according to claim 28, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 30. Thepolynucleotide according to claim 4, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 31. Thepolynucleotide according to claim 30, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 32. Thepolynucleotide according to claim 5, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 33. Thepolynucleotide according to claim 32, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 34. Thepolynucleotide according to claim 6, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 35. Thepolynucleotide according to claim 34, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 36. Thepolynucleotide according to claim 7, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide ofmaize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 37. Thepolynucleotide according to claim 36, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 38. Thepolynucleotide according to claim 8, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 39. Thepolynucleotide according to claim 38, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 40. Thepolynucleotide according to claim 9, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide ofmaize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 41. Thepolynucleotide according to claim 40, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 42. Thepolynucleotide according to claim 10, wherein said mutant polypeptideencoded by said polynucleotide comprises a second amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 43. Thepolynucleotide according to claim 42, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 44. Thepolynucleotide according to claim 13, wherein said mutant polypeptideencoded by said polynucleotide comprises a third amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 45. Thepolynucleotide according to claim 44, wherein the amino acid thatreplaces alanine at position 396 is a valine.
 46. The polynucleotideaccording to claim 14, wherein said mutant polypeptide encoded by saidpolynucleotide comprises a third amino acid mutation wherein the alanineamino acid corresponding to position 396 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 47. The polynucleotide according toclaim 46, wherein the amino acid that replaces alanine at position 396is a valine.
 48. The polynucleotide according to claim 15, wherein saidmutant polypeptide encoded by said polynucleotide comprises a thirdamino acid mutation wherein the alanine amino acid corresponding toposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 49. The polynucleotide according to claim 48, wherein the aminoacid that replaces alanine at position 396 is a valine.
 50. Thepolynucleotide according to claim 16, wherein said mutant polypeptideencoded by said polynucleotide comprises a third amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 51. Thepolynucleotide according to claim 50, wherein the amino acid thatreplaces alanine at position 396 is a valine.
 52. The polynucleotideaccording to claim 17, wherein said mutant polypeptide encoded by saidpolynucleotide comprises a third amino acid mutation wherein the alanineamino acid corresponding to position 396 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 53. The polynucleotide according toclaim 52, wherein the amino acid that replaces alanine at position 396is a valine.
 54. The polynucleotide according to claim 18, wherein saidmutant polypeptide encoded by said polynucleotide comprises a thirdamino acid mutation wherein the alanine amino acid corresponding toposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 55. The polynucleotide according to claim 54, wherein the aminoacid that replaces alanine at position 396 is a valine.
 56. Thepolynucleotide according to claim 19, wherein said mutant polypeptideencoded by said polynucleotide comprises a third amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 57. Thepolynucleotide according to claim 56, wherein the amino acid thatreplaces alanine at position 396 is a valine.
 58. The polynucleotideaccording to claim 20, wherein said mutant polypeptide encoded by saidpolynucleotide comprises a third amino acid mutation wherein the alanineamino acid corresponding to position 396 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 59. The polynucleotide according toclaim 58, wherein the amino acid that replaces alanine at position 396is a valine.
 60. The polynucleotide according to claim 21, wherein saidmutant polypeptide encoded by said polynucleotide comprises a thirdamino acid mutation wherein the alanine amino acid corresponding toposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 61. The polynucleotide according to claim 60, wherein the aminoacid that replaces alanine at position 396 is a valine.
 62. Thepolynucleotide according to claim 22, wherein said mutant polypeptideencoded by said polynucleotide comprises a third amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 63. Thepolynucleotide according to claim 62, wherein the amino acid thatreplaces alanine at position 396 is a valine.
 64. The polynucleotideaccording to claim 23, wherein said mutant polypeptide encoded by saidpolynucleotide comprises a third amino acid mutation wherein the alanineamino acid corresponding to position 396 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 65. The polynucleotide according toclaim 64, wherein the amino acid that replaces alanine at position 396is a valine.
 66. The polynucleotide according to claim 24, wherein saidmutant polypeptide encoded by said polynucleotide comprises a thirdamino acid mutation wherein the alanine amino acid corresponding toposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 67. The polynucleotide according to claim 66, wherein the aminoacid that replaces alanine at position 396 is a valine.
 68. Thepolynucleotide according to claim 25, wherein said mutant polypeptideencoded by said polynucleotide comprises a third amino acid mutationwherein the alanine amino acid corresponding to position 396 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 69. Thepolynucleotide according to claim 68, wherein the amino acid thatreplaces alanine at position 396 is a valine.
 70. The polynucleotideaccording to claim 26, wherein said mutant polypeptide encoded by saidpolynucleotide comprises a third amino acid mutation wherein the alanineamino acid corresponding to position 396 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 71. The polynucleotide according toclaim 70, wherein the amino acid that replaces alanine at position 396is a valine.
 72. The polynucleotide according to claim 27, wherein saidmutant polypeptide encoded by said polynucleotide comprises a thirdamino acid mutation wherein the alanine amino acid corresponding toposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 73. The polynucleotide according to claim 72, wherein the aminoacid that replaces alanine at position 396 is a valine.
 74. Thepolynucleotide according to claim 1, wherein said mutant protein encodedby said polynucleotide further comprises an amino acid mutation thatconfers increased seed weight to a plant expressing said polynucleotide.75. The polynucleotide according to claim 74, wherein saidpolynucleotide comprises the Rev6 mutation.
 76. The polynucleotideaccording to claim 74, wherein said polynucleotide encodes a largesubunit AGP enzyme wherein at least one serine residue is insertedbetween the amino acids corresponding to 494 and 495 in the amino acidsequence of wild type large subunit of ADP-glucose pyrophosphorylasepolypeptide of maize of the native AGP enzyme subunit.
 77. Thepolynucleotide according to claim 74, wherein said polynucleotideencodes a large subunit AGP enzyme wherein the amino acid pairtyrosine:serine is inserted between the amino acids corresponding to 494and 495 in the amino acid sequence of wild type large subunit ofADP-glucose pyrophosphorylase polypeptide of maize of the native AGPenzyme subunit.
 78. The polynucleotide according to claim 74, whereinsaid polynucleotide encodes a large subunit AGP enzyme wherein the aminoacid pair serine:tyrosine is inserted between the amino acidscorresponding to 495 and 496 in the amino acid sequence of wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize ofthe native AGP enzyme subunit.
 79. A method for increasing resistance ofa plant to heat stress conditions, said method comprising incorporatinga polynucleotide selected from the group consisting of thepolynucleotide of claim 1, the polynucleotide of claim 2, thepolynucleotide of claim 3, the polynucleotide of claim 4, thepolynucleotide of claim 5, the polynucleotide of claim 6, thepolynucleotide of claim 7, the polynucleotide of claim 8, thepolynucleotide of claim 9, the polynucleotide of claim 10, thepolynucleotide of claim 11, the polynucleotide of claim 12, thepolynucleotide of claim 13, the polynucleotide of claim 14, thepolynucleotide of claim 15, the polynucleotide of claim 16, thepolynucleotide of claim 17, the polynucleotide of claim 18, thepolynucleotide of claim 19, the polynucleotide of claim 20, thepolynucleotide of claim 21, the polynucleotide of claim 22, thepolynucleotide of claim 23, the polynucleotide of claim 24, thepolynucleotide of claim 25, the polynucleotide of claim 26, thepolynucleotide of claim 27, the polynucleotide of claim 28, thepolynucleotide of claim 29, the polynucleotide of claim 30, thepolynucleotide of claim 31, the polynucleotide of claim 32, thepolynucleotide of claim 33, the polynucleotide of claim 34, thepolynucleotide of claim 35, the polynucleotide of claim 36, thepolynucleotide of claim 37, the polynucleotide of claim 38, thepolynucleotide of claim 39, the polynucleotide of claim 40, thepolynucleotide of claim 41, the polynucleotide of claim 42, thepolynucleotide of claim 43, the polynucleotide of claim 44, thepolynucleotide of claim 45, the polynucleotide of claim 46, thepolynucleotide of claim 47, the polynucleotide of claim 48, thepolynucleotide of claim 49, the polynucleotide of claim 50, thepolynucleotide of claim 51, the polynucleotide of claim 52, thepolynucleotide of claim 53, the polynucleotide of claim 54, thepolynucleotide of claim 55, the polynucleotide of claim 56, thepolynucleotide of claim 57, the polynucleotide of claim 58, thepolynucleotide of claim 59, the polynucleotide of claim 60 , thepolynucleotide of claim 61, the polynucleotide of claim 62, thepolynucleotide of claim 63, the polynucleotide of claim 64, thepolynucleotide of claim 65, the polynucleotide of claim 66, thepolynucleotide of claim 67, the polynucleotide of claim 68, thepolynucleotide of claim 69, the polynucleotide of claim 70, thepolynucleotide of claim 71, the polynucleotide of claim 72, and thepolynucleotide of claim 73 in said plant and expressing the proteinencoded by said polynucleotide.
 80. The method according to claim 79,wherein said plant is a monocotyledonous plant.
 81. The method accordingto claim 80, wherein said monocotyledonous plant is selected from thegroup consisting of rice, wheat, barley, oats, sorghum, maize, lilies,and millet.
 82. The method according to claim 79, wherein said plant isZea mays.
 83. The method according to claim 79, wherein said plant is adicotyledonous plant.
 84. The method according to claim 83, wherein saiddicotyledonous plant is selected from the group consisting of peas,alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass,soybean, tobacco, potato, sweet potato, radish, cabbage, rape, appletrees, and lettuce.
 85. A plant or plant tissue comprising apolynucleotide selected from the group consisting of the polynucleotideof claim 1, the polynucleotide of claim 2, the polynucleotide of claim3, the polynucleotide of claim 4, the polynucleotide of claim 5, thepolynucleotide of claim 6, the polynucleotide of claim 7, thepolynucleotide of claim 8, the polynucleotide of claim 9, thepolynucleotide of claim 10, the polynucleotide of claim 11, thepolynucleotide of claim 12, the polynucleotide of claim 13, thepolynucleotide of claim 14, the polynucleotide of claim 15, thepolynucleotide of claim 16, the polynucleotide of claim 17, thepolynucleotide of claim 18, the polynucleotide of claim 19, thepolynucleotide of claim 20, the polynucleotide of claim 21, thepolynucleotide of claim 22, the polynucleotide of claim 23, thepolynucleotide of claim 24, the polynucleotide of claim 25, thepolynucleotide of claim 26, the polynucleotide of claim 27, thepolynucleotide of claim 28, the polynucleotide of claim 29, thepolynucleotide of claim 30, the polynucleotide of claim 31, thepolynucleotide of claim 32, the polynucleotide of claim 33, thepolynucleotide of claim 34, the polynucleotide of claim 35, thepolynucleotide of claim 36, the polynucleotide of claim 37, thepolynucleotide of claim 38, the polynucleotide of claim 39, thepolynucleotide of claim 40, the polynucleotide of claim 41, thepolynucleotide of claim 42, the polynucleotide of claim 43, thepolynucleotide of claim 44, the polynucleotide of claim 45, thepolynucleotide of claim 46, the polynucleotide of claim 47, thepolynucleotide of claim 48, the polynucleotide of claim 49, thepolynucleotide of claim 50, the polynucleotide of claim 51, thepolynucleotide of claim 52, the polynucleotide of claim 53, thepolynucleotide of claim 54, the polynucleotide of claim 55, thepolynucleotide of claim 56, the polynucleotide of claim 57, thepolynucleotide of claim 58, the polynucleotide of claim 59, thepolynucleotide of claim 60, the polynucleotide of claim 61, thepolynucleotide of claim 62, the polynucleotide of claim 63, thepolynucleotide of claim 64, the polynucleotide of claim 65, thepolynucleotide of claim 66, the polynucleotide of claim 67, thepolynucleotide of claim 68, the polynucleotide of claim 69, thepolynucleotide of claim 70, the polynucleotide of claim 71, thepolynucleotide of claim 72, and the polynucleotide of claim
 73. 86. Theplant or plant tissue according to claim 85, wherein said plant or planttissue is monocotyledonous.
 87. The plant or plant tissue according toclaim 86, wherein said monocotyledonous plant or plant tissue isselected from the group consisting of rice, wheat, barley, oats,sorghum, maize, lilies, and millet.
 88. The plant or plant tissueaccording to claim 85, wherein said plant is Zea mays or said planttissue is from Zea mays.
 89. The plant or plant tissue according toclaim 85, wherein said plant or plant tissue is dicotyledonous.
 90. Theplant or plant tissue according to claim 89, wherein said dicotyledonousplant or plant tissue is selected from the group consisting of peas,alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass,soybean, tobacco, potato, sweet potato, radish, cabbage, rape, appletrees, and lettuce.
 91. The plant tissue according to claim 85, whereinsaid plant tissue is a seed.
 92. A mutant starch biosynthesis proteinencoded by the polynucleotide of claim
 1. 93. A method for increasing acharacteristic of a plant selected from the group consisting of seednumber, plant biomass, Harvest Index, flag leaf weight, seed heads, andtotal seed weight, said method comprising incorporating thepolynucleotide of claim 75 into the genome of said plant and expressingthe protein encoded by said polynucleotide molecule.